Carbohydrates are ubiquitous throughout the plant and animal kingdoms. The structures are known to play a number of important roles in many biological processes. For example, carbohydrates are involved in intercellular recognition in mammalian cells. In fungi and plants, carbohydrates are an important structural component in cell walls. Carbohydrates are typically synthesized by enzymes such as glycosyltransferases, which are a group of enzymes that transfer a monosaccharide from an activated sugar nucleotide to acceptor oligosaccharides found on glycoproteins, glycolipids or polysaccharides. Because of the importance of glycosylation in biological systems, it is highly desirable to develop efficient inhibitors for glycosyltransferases and other enzymes involved in the metabolism of carbohydrates. While there has been increasing attention placed on development of glycosyltransferase inhibitors in recent years, there are as yet no glycosyltransferase inhibitors reported that match the characteristics desired for a therapeutic compound.
Most of the work done on glycosyltransferases to date has focused on hydrophilic analogs of the donor and acceptor substrates of these enzymes. (Hashimoto et al. J Org Chem 62:1914-1915 (1997); Hashimoto et al. J Synth Org Ch Japan 55, 325-333 (1997); Muller et al. Angewandte Chemie-International Edition 37: 2893-2897 (1998); Amann et al. Chemistry-A European Journal 4:1106-1115 (1998); Murray et al. Biochemistry 36, 823-831 (1997); Kim et al. J Am Chem Soc 121:5829-5830 (1999), Schmidt et al. Bioorg Med Chem 3, 1747-1750 (1993); Miura et al. Bioorg Med Chem 6, 1481-1489 (1998); and Palcic et al. J Biol Chem 264, 17174-17181 (1989)). The best inhibitors are generally in the μM range, but inhibitors of up to 14 nM have been obtained for a sialyltransferase(Muller et al., supra). Typically, the inhibitors are negatively charged, and therefore unlikely to be orally available unless a suitable prodrug form is identified.
Most inhibitors based on acceptor substrates are synthetic di- or trisaccharide acceptors that bind to the enzyme, but the hydroxyl group to which the transfer normally occurs has been removed (deoxy-) or substituted (e.g. amino group) Kajihara et al. Carbohydr Res 247:179-193 (1993); Stults et al. Glycobiology 9:661-668 (1999); Lu et al. Bioorg Med Chem 4, 2011-2022 (1996); Lowary et al. Carbohydr Res 251:33-67 (1994); Khan et al. J Biol Chem 268:2468-2473 (1993). In general the Ki values of the inhibitors are in the range of the Km value of the acceptor substrates that they replace. However Ki values on the order of 10 μM have been reported for an α-galactosyltransferase (Lowary et al., supra) and for N-acetylglucosaminyltransferase V (Khan et al., supra).
As a class, prior art oligosaccharide based inhibitors are not expected to cross cell membranes and would be considered poor candidates for a therapeutic. However, several groups have demonstrated that disaccharide acceptor substrates suitably modified with hydrophobic aglycons and/or acetyl esters readily enter cells, reach the Golgi compartment. Terminal glycosylation of cell surface glycoproteins can be thus be inhibited due to competitive glycosylation of the disaccharide substrates which are then secreted (Neville et al. Biochem J:307, 791-797 (1995); Kuan et al. J Biol Chem 264:19271-19277 (1989); Sarkar et al. Proc Natl Acad Sci USA 92:3323-3327 (1995); Sarkar et al. J Biol Chem 272:25608-25616 (1997)).
Other compounds such as N-butyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGNJ) have been shown to be inhibitors of glucosylceramide synthetase. N-butyldeoxynojirimycin is better known as a glucosidase inhibitor, but was found to inhibit the enzyme that initiates the synthesis of glucosylceramides by attaching glucose to ceramide (Platt et al. J Biol Chem 269:8362-8365 (1994) and Platt et al. J Biol Chem 269:27108-27114 (1994). NB-DNJ has been demonstrated to reduce glycolipid synthesis in mice (Platt et al. J Biol Chem 272:19365-19372 (1997) Jeyakumar et al. Proc Natl Acad Sci USA 96:6388-6393 (1999); and Andersson et al. Biochem Pharmacol 59:821-829 (2000)).
Based on the above, it is clear that compounds that specifically modulate the activity of particular glycosyltransferases can be useful to control of number of biological processes. Therefore, a need exists for highly efficient inhibitors of glycosyltransferases. The present invention fulfills this and other needs.